The goal of this project is to elucidate the mechanism of inhibition of N-linked mannan synthesis caused by inositol starvation in Saccharomyces cerevisiae. Our hypothesis is that active mannan synthesis is dependent on some form of inositol as a co-factor for the activity of one of the UDP-N-acetyglucosamine transferases. In previous studies, we have found that a rapid decline in the rate of synthesis of phosphatidylinositol and total mannan occurs when inositol auxotrophs of yeast are starved for inositol. The UDP-N-acetylglucosamine transferases, the first enzymes in N-linked mannan synthesis, are greatly reduced in activity while the mannan synthetase, an enzyme complex which completes synthesis in the mannan polymer, has relatively normal activity. We will determine which transferase is blocked by inositol starvation as well as the reason for the loss in activity using an in vitro assay system. We will also correlate in vivo changes in dolichol-linked oligosaccharide intermediates and N-linked mannan synthesis with our in vitro findings. This work will lead to a better understanding of glycoprotein synthesis in yeast in terms of the mannan biosynthetic pathway, the coordination of cell wall and cell membrane synthesis, and the role of inositol-containing lipids and compounds in the synthesis of mannan. The mannan-containing glycoproteins in yeast are similar to those found in higher, eukaryotic organisms.